Journal: PLoS ONE

Article Title: Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

doi: 10.1371/journal.pone.0100633

Figure Lengend Snippet: 10 −7 M estradiol (black bars) increased FUT-4, -5 and -6 mRNA expression compared with vehicle control (white bars). Data are expressed as A) absolute change in C t value normalized to HPRT1 and B) fold increase in mRNA expression over vehicle control normalized using HPRT1. Values shown are mean ± SEM of 4 different donors performed in 3 biological replicates. Total fucose sugar residues in the cytoplasmic protein fractions of ALI cultures were determined by lectin binding assays. Fucose residues were significantly increased with 10 −7 M estradiol using fucose binding lectins, C-D) AAA, E-F) LTA and G-H) UEA-1. D, F, and H are densitometric quantifications of C, E, and G, respectively. The intensity of all bands in each lane in detecting total fucose residues were quantified using the software program Image J and normalized to total protein loaded per lane in milligrams. Data is expressed as fold increase over vehicle control. Values shown are mean ± SEM of experiments performed with N = 4 donors. * P<0.05, ** P<0.01 compared against vehicle control. Non-parametric t-tests were used in all statistical analyses.

Article Snippet: Membranes were incubated with primary antibodies against ER-α, ER-β, NFATc1, MUC5AC, or (LTA-lotus tetragonolobus asparagus pea: H1601-1, AAA-anguilla anguilla lectin from fresh water eel: H4901-1, and UEA-1-ulex europaeus lectin from gorse: H2201-1) lectins (EY Laboratories Inc., San Mateo, CA) followed by incubation of secondary HRP-conjugated anti-rabbit, anti-mouse and beta-actin antibodies for 1 h at room temperature, and visualized using an enhanced chemiluminescence substrate (Thermo Scientific, Ontario, CAN) and a Chemigenius imaging system (Syngene, Cambridge, UK).

Techniques: Expressing, Control, Binding Assay, Software

Journal: Scientific Reports

Article Title: Neu-medullocytes, sialidase-positive B cells in the thymus, express autoimmune regulator (AIRE)

doi: 10.1038/s41598-018-37225-y

Figure Lengend Snippet: X-NANA, UEA-1 or MHC class II, and AIRE staining of isolated thymic stromal cells. These cells were prepared from three thymuses (C57BL/6 male, 6 W) by enzyme digestion, and the epithelial rich fraction (E2) was used. Image groups ( A – E ) and ( F – J ) are stained with X-NANA, FITC-UEA-1, and anti-AIRE coupled with Rhodamine Red TM -X secondary antibody but shown different fields. Image group ( K – O ) was stained with FITC-anti-MHC class II instead of FITC-UEA-1. ( A , F and K ), X-NANA (blue); ( B , G and L ), UEA-1 or MHC class II (green); ( C , H and M ), AIRE (red). ( D , I and N ) are the merged images of the three colors; ( E , J and O ) are DIC images. Scale bars in A, F and K are 5 μm. Cell 1 was X-NANA, UEA-1 and AIRE positive cell, cell 2 was X-NANA negative, UEA-1 and AIRE positive cell, cell 3 was X-NANA and AIRE positive, but UEA-1 negative cell, cell 4 was X-NANA and AIRE negative but UEA-1 positive, and cell 5 was X-NANA, MHC class II and AIRE positive. Cell (1) expressed weaker X-NANA sialidase activity than that of cell 1.

Article Snippet: UEA-1 ( Ulex europaeus agglutinin, manufactured by J-Oil Mills, Inc.) was purchased from Cosmo Bio Co. (Tokyo, Japan) and labeled with FITC, according to the previously reported method .

Techniques: Staining, Isolation, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Nanoemulsion adjuvantation strategy of tumor-associated antigen therapy rephrases mucosal and immunotherapeutic signatures following intranasal vaccination

doi: 10.1136/jitc-2020-001022

Figure Lengend Snippet: Tailoring emulsified particles to monodisperse nanosized distribution rephrases mucosal signatures following intranasal vaccination. (A) Schematic illustration of the monodisperse nanoemulsion enables the antigens to pass through the mucosal epithelium and facilitate the transportation of antigens into lymphoid tissues. (B) Microarray analysis of transcription profiles induced by emulsified particles 20 hours after administration. Genes with a fold change ≥1.5 and p<0.05 compared with the PBS control. (C) Membranous (M) cell emergence and natural killer (NK) cell trafficking in nasal mucosa. Nasal mucosal tissues were harvested and phenotyped by immunohistochemical (IHC) staining. The brown signal around the blue nucleus indicates UEA-1+ and CD335+ cells, respectively (magnification, 400×). APCs, antigen-presenting cells. PBS, phosphate-buffered saline.

Article Snippet: The primary antibodies, anti-CD3, anti-CD4, anti-CD8, anti-CD335 NKp46 (BioLegend) and anti-UEA-1 (MBL, IL, USA), were applied, and the sections were then incubated at room temperature for 1 hour.

Techniques: Microarray, Immunohistochemical staining, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Immunohistochemical analysis for the specificity of NKM 16–2-4. (A) Immunohistochemical analysis of PPs revealed that NKM 16–2-4 specifically reacted with UEA-1–positive M cells (red arrows), but not UEA-1–positive goblet cells (yellow arrowheads). (B) Electronmicroscopic analysis revealed that typical M cells, which had short and irregular microvilli and pocket structures containing lymphocytes and/or monocytes, specifically reacted with NKM 16–2-4. Positive reactions are shown by gold particles (18 nm). IEC, intestinal epithelial cell. (C) Whole-mount staining of PPs and villous epithelium demonstrated that, in addition to PP-associated M cells, UEA-1–positive villous M cells were specifically recognized by NKM 16–2-4. Bars, 50 μm.

Article Snippet: For blocking analysis, 500 ng/ml Alexa Fluor 647–conjugated NKM 16–2-4 or 500 ng/ml PE-conjugated UEA-1 was first pretreated with 0.5 M α-L-fucose (Wako).

Techniques: Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Development of an M cell–targeted mucosal vaccine with NKM 16–2-4. (A) FITC-conjugated NKM 16–2-4, but not FITC-conjugated control rat IgG, was specifically attached to the apical surfaces of M cells in FAE of PPs within 10 min of inoculation in an intestinal loop assay. The NKM 16–2-4 was subsequently taken up into the cytoplasmic regions of M cells within 30 min and reached the basal membrane of M cells within 4 h. Bars, 10 μm. (B) TT conjugated with NKM 16–2-4 effectively induced high-level, TT-specific serum IgG and fecal IgA responses, unlike TT conjugated with control rat IgG or UEA-1. Furthermore, the levels were superior to those in mice immunized with 10 times the amount of noncoupled TT (500 μg). *, P < 0.01, Tukey's t test. (C) BT-conjugated NKM 16–2-4, but not BT-conjugated control rat IgG, induced brisk botulinum toxin–specific serum IgG and fecal IgA responses. (D) Mice orally immunized with BT-conjugated NKM 16–2-4 were protected from an i.p. challenge with 10,000× LD 50 type A botulinum toxin. Data are expressed as the mean ± the SD.

Article Snippet: For blocking analysis, 500 ng/ml Alexa Fluor 647–conjugated NKM 16–2-4 or 500 ng/ml PE-conjugated UEA-1 was first pretreated with 0.5 M α-L-fucose (Wako).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Effective uptake and universality of the M cell–targeted mucosal vaccine. (A) Immunocytochemical analysis showed that an M cell–targeted OVA vaccine composed of Alexa Fluor 647–conjugated OVA, FITC-conjugated avidin, and NKM 16–2-4 specifically reacted with isolated UEA-1–positive M cells. (B) In an intestinal loop assay, the M cell-targeted OVA specifically attached to the apical surfaces of M cells (red arrows) and was immediately taken up into the cytoplasmic regions of M cells. Bars, 10 μm. (C) Orally administered OVA-conjugated NKM 16–2-4 effectively induced an OVA-specific serum IgG response, whereas an OVA-conjugated control rat IgG did not. Data are expressed as the mean ± the SD.

Article Snippet: For blocking analysis, 500 ng/ml Alexa Fluor 647–conjugated NKM 16–2-4 or 500 ng/ml PE-conjugated UEA-1 was first pretreated with 0.5 M α-L-fucose (Wako).

Techniques: Avidin-Biotin Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Identification of the antigen recognized by NKM 16–2-4. (A) Immunoprecipitation and Western blot analysis with NKM 16–2-4 were performed with an M cell lysate. 4 major bands (3 bands >250 kD and 1 band of ∼150 kD) were precipitated. A subsequent LC-MS/MS analysis identified the three top bands as maltase glucoamylase and the bottom band as alanyl (membrane) aminopeptidase. (B) Lectin blot analysis performed after immunoprecipitation with NKM 16–2-4 showed that the precipitated antigens were all recognized by UEA-1. (C) mFUT1 and mFUT2 genes were transfected into original CHO cells and CHO-derived mutant lines (Lec1, Lec2, and Lec8 cells) with a pIRES2-EGFP expression system, and the specificity of NKM 16–2-4 and UEA-1 for EGFP-expressing transfectants was analyzed. NKM 16–2-4 and UEA-1 specifically reacted with mFUT1- or mFUT2-expressing original CHO cells. The reactivity of NKM 16–2-4 but not UEA-1 to mFUT1- or mFUT2-expressing Lec2 cells was enhanced compared with that to mFUT1- or mFUT2-expressing CHO cells. On the other hand, mFUT1- or mFUT2-expressing Lec1 or Lec8 cells were not recognized at all by NKM 16–2-4.

Article Snippet: For blocking analysis, 500 ng/ml Alexa Fluor 647–conjugated NKM 16–2-4 or 500 ng/ml PE-conjugated UEA-1 was first pretreated with 0.5 M α-L-fucose (Wako).

Techniques: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Derivative Assay, Mutagenesis, Expressing